New Lipophilic Hydroxamates as Promising Trypanocidal Agents: Design, Synthesis, SAR, and Conformational Behavior Studies

A series of novel hydroxamic acid derivatives was designed and synthesized, and their growth inhibitory activity against bloodstream form Trypanosoma brucei was evaluated. These compounds are based on conformationally constrained, lipophilic, spiro carbocyclic 2,6-diketopiperazine (2,6-DKP) scaffolds and bear a side pharmacophoric functionality that contains an acetohydroxamic acid moiety (CH2CONHOH) linked with the imidic nitrogen atom of the 2,6-DKP ring via an acetamido portion [CH2CON(R), R = H, CH3]. Most of these analogues were active in the midnanomolar to low micromolar range against T. brucei. (S)-Isobutyl- or (S)-benzyl-substitution on the methylene carbon located between the amine nitrogen atom and carbonyl of the 2,6-DKP ring was studied. The effect of the methyl-substitution on the nitrogen atom of the acetamido portion in the side pharmacophoric functionality was also examined. Compounds 22 and 23, bearing an isobutyl- or benzyl-substituent, respectively, and concurrently a methyl-substituent, were found to be the most potent hydroxamates of this series (IC50 = 34 and 53 nM, respectively). Both had promising selectivity over the parasite compared to mammalian cells (SI = 940 and 470, respectively). Moreover, an E/Z conformational behavior study on hydroxamic acid 18 and its methyl-substituted counterpart 21 was undertaken using NMR spectroscopy and theoretical calculations.

H uman African trypanosomiasis (HAT or sleeping sickness) results in significant morbidity and suffering.It is caused by protozoan parasites of the Trypanosoma brucei species complex, that are transmitted by the tsetse fly and can infect both humans and animals, causing HAT in man and nagana in cattle.Over recent years, a combination of public health measures have considerably reduced the incidence of HAT, although the potential for epidemic outbreaks remains a concern. 1 In addition, nagana has been an obstruction to the economic development of rural sub-Saharan Africa and a stumbling block to increased production of livestock and agricultural output.The Food and Agriculture Organisation (FAO) estimates that Africa loses US$ 1.5 billion annually in income from agriculture as a result of trypanosomiasis. 2 In humans, the disease develops in two stages.Patients with early stage disease present with nonspecific symptoms such as fever and weakness.They transition to stage 2 HAT when the parasite crosses the blood brain barrier (BBB).This occurs weeks to years after the initial infection, with the patient developing neurological and psychiatric symptoms such as confusion, lethargy, and convulsions.If left untreated, stage 2 disease is generally fatal.
Due to antigenic variation by the parasite, a vaccine against HAT is unlikely.Drugs that have been used for the treatment of the disease are characterized by high toxicity, low efficacy, high cost and increasing parasitic resistance. 3,4Recent drug discovery efforts for HAT have led to the identification of pafuramidine, fexinidazole, and acoziborole. 5Among these, fexinidazole is the only per os bioavailable drug recently approved for use against both stages of T. b. gambiense HAT. 6espite being a distinct improvement, the treatment regimen involves 10 daily doses under medical supervision, and relapses and toxicity have been reported. 7Concurrently, acoziborole has been under clinical evaluation, while the development of pafuramidine has been halted due to adverse renal effects. 8,9hus, additional safe and effective therapeutic agents are needed for the treatment of this devastating parasitic disease, as well as improved drugs for the veterinary sector.
In our previous work, we have reported on a series of spiro carbocyclic 2,6-diketopiperazine-1-acetohydroxamic acid derivatives (Figure 1, compounds 1a-g, 2, 3, 4a-d, 4f, 5a, 5b, 5d) with T. brucei growth inhibitory potencies in the low nanomolar to submicromolar range without significant toxicity to mammalian cells. 10,11These compounds were based on conformationally constrained lipophilic spiro carbocyclic 2,6-DKP scaffold molecules which were modified by introducing an acetohydroxamic acid moiety (CH 2 CONHOH) into their imidic nitrogen.In this way, the combination of a lipophilic spiro carbocyclic 2,6-DKP scaffold and acetohydroxamic acid moiety in one unified chemical entity resulted in potent trypanocidal agents.Structure−activity relationship (SAR) studies showed that the hydroxamic acid group (CONHOH) in these compounds is of vital importance to their trypanocidal activity, since replacement of this group by an amide (CONH 2 ), a hydrazide (CONHNH 2 ), an O-methyl hydroxamate (CONHOCH 3 ) or a carboxylic acid (COOH) group resulted in inactive compounds. 10On this basis, we hypothesized that these hydroxamates could act by inhibiting essential parasite metalloenzymes, such as members of the iron superoxide dismutase repertoire 12 or the Trypanosome Alternative Oxidase (TAO), 13 through the metal-ion binding action of the hydroxamic acid group (via its Z(cis)conformation) in the catalytic site.As also shown by the SAR study of this hydroxamate series, molecules containing a benzyl substituent at the position adjacent to the amine nitrogen of the 2,6-DKP ring proved to be the most potent antitrypanosomal agents within this series, with single nanomolar or low midnanomolar IC 50 values.
With the aim of developing hydroxamate compounds of improved potency and selectivity against T. brucei, we subsequently modified the spiro carbocyclic 2,6-DKP core structure by replacing the spiro-linked carbocyclic component with an alkyl and an aryl substituent.This modification led to a novel series of acetohydroxamic acid derivatives (Figure 1,  ,11 and 3-alkyl-3-aryl 2,6-diketopiperazine-1-acetohydroxamic acids 6−17 14 reported previously, along with structures of the new hydroxamic acid derivatives 18−28 based on spiro carbocyclic 2,6-diketopiperazine scaffolds.compounds 6-17) based on conformationally non-constrained 3-alkyl-3-aryl-2,6-DKP scaffolds.The generated hydroxamates 6-17 were potent T. brucei growth inhibitors with submicromolar to low micromolar activities.Additionally, the most active of these compounds had promising selectivity over the parasite with respect to mammalian cells. 14SAR studies demonstrated that compounds bearing a methyl, n-propyl or nbutyl substituent at the N(4)-position of the 2,6-DKP ring portion exhibited the highest activity against bloodstream form T. brucei.
In a search for more potent hydroxamic acid−based trypanocidal agents, and to identify the SAR features that may prove useful in future drug discovery efforts, we designed and synthesized a novel series of hydroxamic acid derivatives (Figure 1, compounds 18-28) as T. brucei growth inhibitors.These compounds are based on spiro carbocyclic 2,6-DKP scaffolds and characterized by the presence of an acetamido portion (−CH 2 CONR-) between the imidic nitrogen of the spiro carbocyclic 2,6-DKP scaffold and the acetohydroxamic acid moiety (−CH 2 CONHOH).This modification was hoped to lead to an enhanced enzymic metal ion-hydroxamic acid complex stabilization, due to additional interactions between the acetamido portion (−CH 2 CONR-) and sites within the catalytic region of the biological target(s).More specifically, elongating the acetohydroxamic arm with an additional acetamido group could provide more donor atoms, thus improving metal chelation.Furthermore, substituting the secamide for a tert-amide should enhance the lipophilicity of the ligand while also forcing a Z-conformation (which was proven beneficial in our previous study) due to steric hindrance.Based on this assumption, compounds 18, 24 and 27 (Figure 1, R=R′=H) constituted the molecular templates for further SAR studies.We first examined the effect of the (S)-isobutyl-or (S)benzyl-substitution on the methylene carbon, as the (S)enantiomer was shown in our previous studies to be more potent than the (R)-, 10,11 located between the amine nitrogen atom and the carbonyl of the 2,6-DKP ring (Figure 1, compounds 19, 20, 25 and 28).We subsequently studied the methyl substitution on the nitrogen atom of the acetamido portion (−CH 2 CONH−) in the side pharmacophoric 2-   and 26).The trypanocidal activity of the newly synthesized compounds was assessed against bloodstream form T. brucei in vitro.In addition, the most active compounds were examined for their toxicity to mammalian cells, using the rat skeletal myoblast L6 cell line.
According to the systematic conformational search for compound 18, the conformation of minimum energy is the ZZ (Figure 2 − the nomenclature convention followed is first to address the conformation of the amide and second of the hydroxamic acid group).The conformer is in agreement with characteristic observations in the NOESY spectra which assign the peaks of the major conformer in the 1 H NMR spectrum to NMR for Z and E conformers can be detected under standard conditions.In addition, the presence of the methyl group could prohibit the acetamidoacetohydroxamic acid residue to approach too close to the adamantane ring, thus further enabling an EE conformation.
The coordination scans of the two dihedrals were conducted on a minimized energy structure of the respective compounds (0°-Figures S4, S3, SI) initially one angle at a time while the other remained frozen (Figure 3; Table S2, SI), and in combination for both simultaneously (Figures S1, S2, SI; Table S2, SI).In 18 1_1 the relative ΔU 18_1 = 23.55 kcal/mol between the global maximum and global minimum, while for 2_1 it is ΔU 18_2 = 15.62 kcal/mol.The combination scan indicates that global minimum energy is reached when 1_1 is 360°and 2_1 is 5°, while the relative ΔU 18_C = 35.6 kcal/mol compared to the global maximum (Figure S1, SI; Table S2, SI).The respective values for 21 are ΔU 21_1 = 19.94kcal/mol, ΔU 21_2 = 15.12 kcal/mol and ΔU 21_C = 31.6kcal/mol, with global minimum energy reached in the combination scan when 1_1 is 0°and 2_1 is 360°(Figure S2, SI; Table S2, SI).Comparing the two compounds, we observe that 18 has larger energy differences with ΔΔU 1 = 3.62 kcal/mol, ΔΔU 2 = 0.50 kcal/mol and ΔΔU C = 4.0 kcal/mol.
The structural motif that we can extrapolate from the systematic conformational search of the two compounds to resolve the NOE signals can be summarized as follows: (i) when the hydroxamic acid group adheres to E conformation, the −OH should approach the α-CH 2 of the hydroxamic carbonyl; (ii) when the amide adheres to E conformation, the α-CH 2 of the hydroxamic carbonyl should approach the α-CH 2 of the amide carbonyl in both compounds: 18 (R = H) and 21 (R = CH 3 ).In addition, from the results of the conformational search for the two compounds, we can see that the relative energy differences (ΔU) of the different rotamers in 18 are significantly higher than for 21.Nonetheless, since 21 has an additional methyl group on the amide nitrogen which adds steric hindrance to the system, its minimum energy conformation is in a higher absolute energy level (an approach of this is expressed by the force field energy U).Thus, 21 has lower energy barriers between its rotamers, although its minimum energy conformation is situated in a higher energy level, while in a reverse manner, 18 has higher energy barriers between its rotamers, and its minimum energy conformation is situated in a lower energy level.This profiling suggests that it should be energetically easier for 21 to populate the rotamers of higher energy than for 18.This is further verified by the results of the dihedral coordination scans, where again the relative energy differences are significantly higher in 18.An examination of the output structures of the coordination scans illustrates that, although more sterically hindered, the dihedral 1_1 of 21 is more free to rotate than that of its counterpart 18 (Figures S10−S13, SI).The secondary amide group in 18 can tautomerize toward the respective imidic acid, whereas the tertiary amide in 21 is not eligible for tautomerization, while both have access to the respective mesomeric zwitterion structure (Scheme 2).As a result, the amide bond in 18 is more rigid than that in 21, as in the former its character is more strongly shifted toward a double bond relative to the latter.Therefore, despite the steric hindrance, the amide bond in 21 is more prone to rotate than that in 18, providing an explanation for the energy differences of the coordination scans, as well as for the energy barriers between the rotamers derived from the conformational search.
Biological Activity.The new hydroxamic acid derivatives 18−28 were tested against bloodstream-form T. brucei in vitro.The results are presented in Table 1, and are expressed as IC 50 and IC 90 values.Compounds 19-23, 25 and 28 displayed potent trypanocidal activities with midnanomolar to low micromolar IC 50 values (0.034−1.65 μM), while having low toxicity against mammalian cells (rat skeletal myoblast L6 cells) (Table 1).In this new series of hydroxamates 18-28, the SAR study revealed the following.
Insertion of an acetamido portion (−CH 2 CONH−) between the spiro adamantane 2,6-DKP scaffold and acetohydroxamate pharmacophoric group (−CH 2 CONHOH) in compound 1a (IC 50 = 0.09 μM), leading to compound 18 (IC 50 = 25 μM), caused a 278-fold loss in potency.The same modification on the cyclooctane-and cycloheptane-based acetohydroxamic acid analogues 4a (IC 50 = 0.30 μM) and 5a (IC 50 = 1.87 μM), giving the respective compounds 24 and 27, also led to substantial loss of activity (compare compounds 4a vs 24 and 5a vs 27).However, attachment of the hydrophobic isobutyl substituent to the methylene carbon of the 2,6-DKP ring in the parent compounds 18, 24 and 27, resulting in the (S)-enantiomers of the corresponding isobutyl-substituted analogues 19, 25 and 28, greatly enhanced activity against T. brucei.Indeed, compounds 19, 25 and 28 were 62, > 252 and >18 times more potent than the unsubstituted parents 18, 24 and 27, respectively, with IC 50 values in the submicromolar (19) or low micromolar (25, 28) range (Table 1).A similar trend was observed when the bulky hydrophobic benzyl substituent was incorporated into the same carbon of the 2,6-DKP ring in the adamantane-based parent 18.The resulting benzyl substituted analogue 20 [(S)-enantiomer] showed a 20fold increase in potency relative to the unsubstituted parent 18, with a low micromolar IC 50 value (Table 1).The large increase in potency for 19, 20, 25 and 28 probably reflects the favorable lipophilic and/or stereoelectronic effects exerted by the bulky isobutyl and benzyl substituents in the target binding site.To further address the impact of the different substitution patterns and residues in lipophilicity, we conducted a modeling calculation of drug-like properties and descriptors using the QikProp module of the Schrodinger platform (Table S3, cmp_qikprop.out).The range of the predicted logP for all compounds spans from −1.9 to 0.4, with the lowest values adhering to the sec-amides 18, 24, 27 and the ring unsubstituted 21, 26 (Table S3).Additionally, the introduction of a methyl substituent to the nitrogen atom of the acetamido portion (−CH 2 CONH−) in the pharmacophoric 2a c e t a m i d o a c e t o h y d r o x a m i c a c i d f u n c t i o n a l i t y Scheme 2. Tautomerization and Resonance Structures of sec-Amides and tert-Amides  (−CH 2 CONHCH 2 CONHOH) of compounds 18-20 and 24 appeared to have a favorable effect on the trypanocidal activity and resulted in a significant potency increase for the corresponding N-methylated analogues 21-23 and 26; they were recorded as being 19, 12, 23 and >8 times more potent than the nonmethylated counterparts 18-20 and 24, respectively.Compounds 21-23 inhibited T. brucei growth at midnanomolar (22, 23) or low micromolar (21) levels.Interestingly, 22 and 23 exhibit the highest predicted values for apparent Caco-2 cell permeability (31 nm/sec and 19 nm/ sec, respectively − Table S3), as well as oral absorption percentage (53% and 51%, respectively − Table S3).Thus, even though the models suggest a low level of lipophilicity for 22 and 23 (logP = −0.1 and 0.2, respectively − Table S3), they are predicted to exhibit significantly better permeability and absorption than the other compounds.In addition, these results might be attributed to the favorable spatial arrangement of the methylated side pharmacophoric functionality [−CH 2 CON(CH 3 )CH 2 CONHOH] for interactions with the active site, probably due to the conformational profile described above.
Importantly, the activity results show that the combination of isobutyl-or benzyl-substitution on the 2,6-DKP ring and the concomitant methylation of the side pharmacophoric moiety in the adamantane-based unsubstituted parent compound 18 drastically increased the antitrypanosomal potency and gave the more active compounds 22 and 23 (IC 50 values: 0.034 and 0.053 μM, respectively).Compounds 22 and 23 appeared to be 735 and 472 times more potent than 18, respectively.While each of the two modifications separately led to less active compounds (19, 20 and 21), both modifications in concert provided the best antitrypanosomal effect.
It is worth noting that the most active compounds (19-23, 25 and 28) were found to have significant selectivity for T. brucei compared to mammalian cells used (SI = 90−940, Table 1), except for 20 (SI = 24).Among them, the adamantanebased hydroxamates 22 and 23, which displayed the most potent trypanocidal activity (IC 50 = 0.034 and 0.053 μM, respectively) proved to be the most parasite-selective compounds (SI = 940 and 470, respectively).Last but not least, QikProp models suggest that our compounds have a property similarity ranging 70−81% compared to marketed drug APIs from a database of 1712 APIs (cmp_qikprop.out),indicating their significant therapeutic potential.

■ CONCLUSION
We have generated a new series of hydroxamic acid derivatives that are based on spiro carbocyclic 2,6-DKP scaffolds.Most of them inhibit the bloodstream-form T. brucei parasite growth with low micromolar to midnanomolar IC 50 values.The modification of the previously reported acetohydroxamic acid derivatives 1a, 4a and 5a (Figure 1) by inserting an acetamido portion (−CH 2 CONH−) between the 2,6-DKP ring and the acetohydroxamate group (−CH 2 CONHOH) resulted in compound 18 with marginal activity, and the inactive compounds 24 and 27.However, compounds 18, 24 and 27 were successfully transformed into the (S)-isobutyl-or (S)benzyl-substituted analogues 19, 20, 25 and 28 that displayed submicromolar (19) or low micromolar (20, 25 and 28) trypanocidal activities by attaching an isobutyl or benzyl substituent at the vicinal position of the basic nitrogen atom in the 2,6-DKP ring.Conversely, methyl-substitution on the nitrogen atom of the acetamido portion (CH 2 CONH−) in the side pharmacophoric hydroxamate functionality (−CH 2 CONHCH 2 CONHOH) of compounds 18-20 and 24 significantly improved trypanocidal potency, as represented by the respective methylated analogues 21-23 and 26.In particular, compounds 22 and 23 [(S)-enantiomers] displayed midnanomolar trypanocidal activities, demonstrating that the isobutyl-or benzyl-substitution on the 2,6-DKP ring in conjunction with methylation of the side pharmacophoric moiety in the adamantane-based unsubstituted parent compound 18 was crucial in terms of activity against bloodstream form T. brucei.Indeed, compounds 22 and 23 exhibited the highest growth inhibitory activity (within the series), with significant selectivity, compared to the mammalian cells.This SAR, taken together with the computational studies conducted on 18 and 21, demonstrate that the freedom of the acetamidoacetohydroxamic arm to rotate is of paramount importance for achieving high ligand potency.
On the basis of these encouraging findings, compounds 22 and 23 were selected as chemical leads for the development of new hydroxamic acid derivatives with optimized antitrypanosomal potency and outstanding selectivity against the parasite, through appropriate substitutions in both the 2,6-DKP portion and side pharmacophoric moiety.
Full detailed report of all QikProp models and predicted values (TXT) Experimental procedures for the synthesis of the target compounds; experimental procedures for the HPLC quantitative determination; experimental procedures for the computational studies; Experimental procedures for the biological evaluation of the compounds; drug-like properties predictions for the tested compounds; copies of NMR spectra of all synthesized compounds (PDF) ■
a c e t a m i d o a c e t o h y d r o x a m i c a c i d f u n c t i o n a l i t y (−CH 2 CONHCH 2 CONHOH) (Figure 1, compounds 21-23 C NMR spectra as E and Z conformers (not assigned) due to the hindered rotation around the C(O)-N(CH 3 ) amide bond.The nonmethylated glycine analogues 47-49, 53, 54, 56 and 57 were subjected to efficient EDCI-HOBt coupling reactions with O-benzylhydroxylamine to afford the corresponding O-benzyl hydroxamates 58-60, 64, 65, 67 and 68 in 57−78% yields.As the EDCI-HOBt coupling reactions of the N-methylated glycine analogues 50-52 and 55 with O-benzylhydroxylamine give products in low yields (<30%), we favored CDI activation coupling in a 48 h reaction time.Thus, we obtained the corresponding methylated O-benzyl hydroxamates 61-63 and 66 in considerably improved yields (64−75%).Removal of the benzyl-protective group in compounds 58-68 by catalytic hydrogenolysis (H 2 / 10% Pd−C) afforded the respective hydroxamic acid analogues 18-28 in high yields (88−97%).

Figure 2 .
Figure 2. Conformation of minimum energy of 18 (left) and 21 (right).Distances in Å between hydrogens supporting NOE signals are marked in violet.The hydrogen of the hydroxamic OH in 18 is not visible due to the angle of the snapshot; both compounds share the same protonation state (cf.Figures S3−S13).

c
Cytotoxicity was determined by establishing the concentration required to inhibit growth of cultured L6 cells by 50% (IC 50 ).Data are the mean of triplicate experiments ± standard error of the mean (SEM).d Selectivity indices (SIs) were calculated as the ratio of the IC 50 for L6 cells and T. brucei.e Data in brackets refer to the respective hydrochloride.ND: Not determined. 10

Table 1 .
17tivity of Hydroxamic Acid Analogues 18-28 Tested against Cultured Bloodstream-form Trypanosoma brucei (pH = 7.4) and Cytotoxicity of the Most Active Compounds against Cultured Rat Skeletal Myoblast L6 Cells (Experimental Details in the SI)17Concentrations required to inhibit growth of T. brucei by 50% and 90%, respectively.b IC 50 and IC 90 data are the mean of triplicate experiments ± standard error of the mean (SEM). a